Introduction: This study investigated the gender differences in reported barriers to participation in after-school physical activity (PA) and related health and socio-behavioural factors in Australian schoolchildren. Methods: 5001 students aged 10 to 16 years completed the health and well-being survey in 2014 indicating that they would like to participate in after-school PA. Negative binomial regression models, stratified by gender, tested the relationship of age, reported health, junk food, participation in leisure PA, TV watching, weight status and socio-economic index for area score (related factors) with the total number of barriers. Results: Girls were more likely to report a greater number of barriers to participation in after-school PA than boys (P <.05). Older age was associated with a higher number of barriers in girls (B(95% CI) = 1.061 (1.032, 1.090)) but not in boys. In both boys and girls, being overweight (boys: very overweight (1.367 (1.081, 1.730)); girls: slightly overweight (1.186 (1.100, 1.278)) or very overweight (1.414 (1.197, 1.667))), compared to students who reported “being about the right weight,” was associated with a greater number of barriers. Schoolchildren who reported less than excellent health status perceived a greater number of barriers to after-school PA (girls: good (1.141 (1.060, 1.228)), fair (1.189 (1.070, 1.321)) and poor health (1.329 (1.093, 1.614)); boys: good health (1.166 (1.0728, 1.267))). Conclusions: There are gender differences in barriers to participation in after-school PA; these should be taken into account when developing programs to increase schoolchildren's after-school PA. So what: The prevalence of physical inactivity in Australian adolescents is staggering. We observed that girls reported a greater number of barriers to participation in after-school PA than boys; and being overweight and reporting poorer overall health were associated with a greater number of barriers. Affordable, gender- and age-specific after-school PA programs suitable for schoolchildren of all body shapes and sizes and all abilities are needed.